The aim of this study was to prepare poly (lactide-co-glycolide) (PLGA) microspheres encapsulating highly concentrated glycyrrhizin (GZ), a hydrophilic drug, and to compare the release characteristics of GZ in in vitro experiments and GZ elimination into bile after subcutaneous administration in rats. The preparation was carried out based on water drying using a (w/o)/w emulsion. The encapsulation rate of GZ in microspheres was 76% when the GZ concentration in the outer water phase was equal to that in the inner water phase for the preparation of (w/o)/w emulsion. The release of GZ from the microspheres showed a biphasic zero-order profile, that is, the behavior boundary was approximately 12 h. The release of GZ from the microspheres at the periods of 0.5-8 h and 12-672 h was 0.18 mg/h and 0.0012 mg/h, respectively. On the other hand, 0.25% of GZ administered (5.0 mg) was eliminated into bile by 12 h, and the bile clearance rate (1.13 ml/h) of GZ after the subcutaneous administration of the microspheres was the same as that (1.13 ml/h) after the administration of GZ solution. From the results, it is suggested that the initial controlled release (0.18 mg/h) of GZ from microspheres may be beneficial for the hepatic bioavailability of GZ.